This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The objectives of this proposal are to address two unknowns about the virulence of the bacterium Pseudomonas aeruginosa. The first objective is to identify the mechanism(s) causing mucoid conversion of clinical isolates from cystic fibrosis patients besides mutations in mucA. The second objective is to determine if the role that the chaperone SpcU plays in the secretion of the Type III exotoxin ExoU is to keep ExoU soluble and stable. ExoU is a potent cytotoxin of P. aeruginosa. The long term goal of this research is to increase our understanding of the mechanisms used by bacteria to cause disease. The hypotheses to be tested are: 1) that defects in factors upstream of MucA or in the RpoN pathway mediate mucoid conversion in mucA+ clinical isolates and (2) that the P. aeruginosa chaperone SpcU acts to keep the toxin ExoU soluble and secretion competent by binding to both its C-terminal membrane localization domain and its N-terminal chaperone binding domain. These hypotheses will be tested by pursuing two specific aims: 1) Identify mechanisms mediating mucoid conversion in mucA+ strains;and 2) Examine the role of Pseudomonas chaperones in promoting the solubility, stability and secretion of exotoxins. For aim 1, we will perform genetic analyses of mucoid mucA+ clinical isolates to determine if recently identified regulators (AlgW and RpoN) are activated in these strains. For aim 2, we will examine the effect of mutations of the C-terminal membrane localization domain and N-terminal chaperone binding domain of ExoU on its interaction with SpcU, and on its solubility, stability and ability to be secreted. This proposal is innovative because it will take advantage of unique properties of the exotoxin ExoU and of our discovery and characterization of additional regulators of mucoid conversion. The proposed research is significant because it is expected to advance our understanding of two virulence mechanism used by P. aeruginosa and other pathogenic bacteria.